The long-term goal of the project is to understand the mechanisms by which cells maintain trinucleotide repeat (TNR) stability. Expansions of TNR sequences, e.g., (CAG)n and (CTG)n, are tightly associated with progression of certain human neurological and neurodegenerative diseases including Huntington's disease (HD) and myotonic dystrophy. However, the mechanisms and factors that promote/prevent TNR expansions are unknown. Because CAG and CTG repeats form thermo- stable hairpins with multiple A-A and T-T mispairs in the hairpin stem, respectively, DNA mismatch repair (MMR) and TNR hairpin repair have been proposed to play major roles in TNR maintenance. Surprisingly, previous studies in transgenic mice suggest that mismatch recognition protein MSH2- MSH3 heterodimer (also called MutS2) promotes (CAG)n expansions by binding to (CAG)n-formed hairpins and inhibiting their repair. Our recent studies have shown that human cells catalyze error-free repair of (CAG)25 and (CTG)25 hairpins in a nick-directed PCNA-dependent manner. The repair targets the nicked strand for incisions at the repeat sequences, followed by repair DNA synthesis using the continuous strand as a template, thereby ensuring TNR stability. However, MutS2 does not inhibit, stimulates (CAG)25 or (CTG)25 hairpin repair. Interestingly, our preliminary studies have shown that cell lines derived from HD patients are defective in (CAG)n hairpin repair, hinting possible pathogenesis for HD. In this application, Specific Aim 1 is to evaluate the model that the MMR system promotes (CAG)n expansion. Human and animal cell lines with or without MutS2 overexpression will be examined for their ability to repair (CAG)n hairpins in vitro and to replicate CAG repeats in vivo. Determination of TNR hairpin repair activity and (CAG)n stability in these cells will clarify if the MMR system is responsible for (CAG)n expansions in human cells. Specific Aim 2 is to further test the hypothesis that hairpin repair defects are associated with TNR diseases, by screening hairpin repair proficiency in cell lines derived from HD patients. Specific Aim 3 is to purify and characterize a protein required for (CAG)n hairpin repair but defective in an HD cell line. A successful completion of the proposed work will provide significant insight into the mechanisms of TNR expansions and the etiology of TNR expansion-associated diseases. PUBLIC HEALTH RELEVANCE: Expansion of simple nucleotide repeats in DNA is the genetic basis for more than 40 human familial neurological, neurodegenerative and neuromuscular disorders, including Huntington's disease, myotonic dystrophy, Friedreich ataxia, and fragile X syndrome. The repeat expansion can occur in any part of a gene and leads to a defective gene product. However, how the repeat units expand and what cellular mechanism(s) prevent such an expansion in normal population are not fully understood. This application is to identify protein components that promote or prevent repeat expansions. A successful completion of the proposed work will provide significant insight into the pathogenesis underlying diseases associated with repeat expansions and approaches to develop effective treatments for the diseases.